Chloroplast transit peptides for efficient targeting of DMO and uses thereof

ABSTRACT

The invention provides for identification and use of certain chloroplast transit peptides for efficient processing and localization of dicamba monooxygenase (DMO) enzyme in transgenic plants. Methods for producing dicamba tolerant plants, methods for controlling weed growth, and methods for producing food, feed, and other products are also provided, as well as seed that confers tolerance to dicamba when it is applied pre- or post-emergence.

This application is a divisional of U.S. Ser. No. 12/914,901, filed Oct.28, 2010, now U.S. Pat. No. 8,084,666, which application is a divisionalof U.S. Ser. No. 11/758,659, filed Jun. 5, 2007, now U.S. Pat. No.7,838,729, which claims the priority of U.S. Provisional PatentApplication 60/891,675, filed Feb. 26, 2007, the disclosures of each ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of plant biotechnology. Moreparticularly, the invention relates to identification and use ofchloroplast transit peptides allowing efficient processing andlocalization of dicamba monooxygenase enzymes in plants.

2. Description of the Related Art

DMO (dicamba monooxygenase) catalyzes the degradation of the herbicidedicamba (3,6-dichloro-o-anisic acid) to non-toxic 3,6-dichlorosalicylicacid (3,6-DCSA) in plants, thus conferring herbicide tolerance. Activityof DMO requires two intermediary proteins for shuttling electrons fromNADH to dicamba, a reductase and a ferredoxin (U.S. Pat. No. 7,022,896;Herman et al., 2005). However dicamba tolerance in transgenic plants hasbeen demonstrated through transformation with DMO alone, indicating thata plant's endogenous reductase and ferredoxin may substitute inshuttling the electrons. The plant ferredoxin that is involved inelectron transfer is localized in the plastids. Thus, in order to obtainefficient performance of DMO and thus improved tolerance to dicamba,there is a need for targeting the DMO to chloroplasts.

In many cases, this targeting may be achieved by the presence of anN-terminal extension, called a chloroplast transit peptide (CTP) orplastid transit peptide. Chromosomal transgenes from bacterial sourcesmust have a sequence encoding a CTP sequence fused to a sequenceencoding an expressed polypeptide if the expressed polypeptide is to becompartmentalized in the plant plastid (e.g. chloroplast). Accordingly,localization of an exogenous polypeptide to a chloroplast is oftenaccomplished by means of operably linking a polynucleotide sequenceencoding a CTP sequence to the 5′ region of a polynucleotide encodingthe exogenous polypeptide. The CTP is removed in a processing stepduring translocation into the plastid. Processing efficiency may,however, be affected by the amino acid sequence of the CTP and nearbysequences at the NH₂ terminus of the peptide.

Weeks et al. (U.S. Pat. No. 7,022,896) describe potential use of a maizecab-m7 signal sequence (see Becker et al., 1992 and PCT WO 97/41228;GenBank Accession No. X53398) and a pea glutathione reductase signalsequence (Creissen et al., 1992 and PCT WO 97/41228) in targeting DMO toplant plastids, but no data on efficiency of processing or targeting isgiven. A pea Rubisco small subunit (RbcS) CTP including a 27 aa sequenceincluding coding sequence for pea Rubisco enzyme small subunit has alsobeen used to target DMO to chloroplasts (e.g. U.S. Prov. Appl. Ser. No.60/811,152). However, it has been found during Western blot analysisthat this pea RbcS CTP generates a correctly processed DMO protein band(˜38 kDa), but also a larger band (˜41 kDa) corresponding to that of DMOand the 27 aa of RbcS coding region. The extra amino acids could impactthe DMO activity adversely. In addition, additional proteins in atransgenic product due to incomplete processing of DMO create regulatoryhurdles and require additional efforts in characterization of theproduct for the purposes of product registration by government agenciesthereby raising the cost of product registration. Thus, there is a needfor identifying CTPs that efficiently generate correctly processed DMO,thereby providing the advantage of full DMO activity as well as ease ofproduct characterization.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a recombinant DNA moleculecomprising a nucleotide sequence encoding a chloroplast transit peptideoperably linked to a nucleotide sequence encoding dicamba monooxygenase,wherein the nucleotide sequence encodes a chloroplast transit peptidecomprising a sequence selected from the group consisting of SEQ ID NOs:1-11. In certain embodiments, the recombinant DNA molecule comprises anucleotide sequence selected from the group consisting of SEQ ID NOs:12-22. In certain embodiments, the recombinant DNA molecule comprises anucleotide sequence encoding dicamba monooxygenase selected from thegroup consisting of SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, and 40.A DNA construct comprising the DNA molecule operably linked to apromoter which is functional in a plant cell is also an aspect of theinvention.

In another aspect, the invention comprises a plant cell transformed witha DNA molecule comprising a nucleotide sequence encoding a chloroplasttransit peptide operably linked to a nucleotide sequence encodingdicamba monooxygenase, wherein the sequence of the chloroplast transitpeptide is selected from the group consisting of SEQ ID NOs: 1-11. Incertain embodiments, the recombinant DNA molecule comprises a nucleotidesequence selected from the group consisting of SEQ ID NOs: 12-22. Incertain embodiments, the DNA molecule comprises a nucleotide sequenceencoding a dicamba monooxygenase selected from the group consisting ofSEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, and 40, wherein the DNAmolecule is operably linked to a promoter which is functional in a plantcell. In particular embodiments the DNA molecule comprises a nucleotidesequence selected from the group consisting of SEQ ID NOs: 23, 25, 27,29, 31, 33, 35, 37, and 39.

In certain embodiments, the plant cell is a dicotyledonous plant cell.In other embodiments, the plant cell is a monocotyledonous plant cell.In particular embodiments, the plant cell is a soybean, cotton, maize,or rapeseed plant cell. The invention also relates to a plant tissueculture comprising such a cell, and to a transgenic seed and to atransgenic plant comprising such cells. In certain embodiments, thetransgenic seed or plant is a dicotyledonous seed or plant. In otherembodiments, the transgenic seed or plant is a monocotyledonous seed orplant. The transgenic seed or plant may be a soybean, cotton, maize orrapeseed seed or plant.

The invention further relates to a method for producing a dicambatolerant plant comprising: introducing a recombinant DNA moleculecomprising a nucleotide sequence encoding a chloroplast transit peptideoperably linked to a nucleotide sequence encoding dicamba monooxygenase,wherein the nucleotide sequence encoding the chloroplast transit peptideis selected from the group consisting of SEQ ID NOs: 12-22, into a plantcell, and regenerating a plant therefrom. In certain embodiments, therecombinant DNA molecule comprises a nucleotide sequence encodingdicamba monooxygenase which is selected from the group consisting of SEQID NOs: 24, 26, 28, 30, 32, 34, 36, 38, and 40. The DNA molecule may beoperably linked to a promoter which is functional in a plant cell. Themethod may further comprise producing a dicamba tolerant plant bycrossing a parent plant with itself or with a second plant, wherein theparent plant and/or the second plant comprises the DNA construct and thedicamba tolerant plant inherits the DNA construct from said parent plantand/or the second plant.

A method for expressing dicamba monooxygenase in a plant cell comprisingoperably linking a selected CTP to a sequence encoding dicambamonooxygenase is a further aspect of the invention.

In another aspect, the invention relates to a method for controllingweed growth in a crop growing environment, comprising: growing such aplant or a seed thereof, and applying to the crop growing environment anamount of dicamba herbicide effective to control weed growth. Thedicamba herbicide may be applied over the top to the crop growingenvironment, whereby the amount of dicamba herbicide does not damagesaid plant of or seed thereof and damages a plant or seed of the samegenotype as such a plant or seed but lacking the construct.

A further aspect of the invention relates to a method for producingfood, feed, or an industrial product comprising:

-   -   a) obtaining a plant comprising a nucleotide sequence encoding a        promoter functional in a plant cell operably linked in the 5′ to        3′ direction to a nucleotide sequence encoding a chloroplast        transit peptide and a nucleotide sequence encoding dicamba        monooxygenase, or a part thereof;    -   b) preparing the food, feed, fiber, or industrial product from        the plant or part thereof.        In certain embodiments of the method, the food or feed is grain,        meal, oil, starch, flour, or protein. In other embodiments of        the method, the industrial product is biofuel, fiber, industrial        chemicals, a pharmaceutical, or nutraceutical.

A dicamba tolerant seed for providing protection against pre emergenceapplication of dicamba comprising a DNA encoding chloroplast transitpeptide operably linked to a DNA encoding dicamba monooxygenase is afurther aspect of the invention. In certain embodiments, the dicambatolerant seed comprises a nucleotide sequence encoding a chloroplasttransit peptide, such as a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 12-22. The dicamba tolerant seed may furthercomprise a nucleotide sequence encoding dicamba monooxygenase selectedfrom the group consisting of SEQ ID NOS: 24, 26, 28, 30, 32, 34, 36, 38,and 40.

Another aspect of the invention relates to a method for improving thestandability of a monocot plant comprising: a) obtaining and growing aplant produced by crossing a parent plant with itself or with a secondplant, wherein the parent plant and/or the second plant comprises theDNA construct and the dicamba tolerant plant inherits the DNA constructfrom said parent plant and/or the second plant; and b) treating theplant with dicamba. In certain embodiments, the plant is a corn plant.In yet other embodiments, standability-related parameters includingbrace root shape, number, length, and/or structure; percent lodging; andyield may be measured.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Use of CTP-DMO constructs for proper processing of DMO andprovision of dicamba tolerance.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, compositions and methods are providedfor expressing and transporting dicamba monooxygenase (DMO) polypeptideswith increased efficiency to chloroplasts in plant cells. Thecompositions and methods of the invention will therefore find use inincreasing the tolerance of plants and cells to the herbicide dicamba.By targeting DMO to chloroplasts with a chloroplast transit peptide(CTP) in particular, improved DMO expression and tolerance to dicambamay be achieved.

Surprisingly, however, the present inventors have discovered thatcertain CTPs do not function well in combination with DMO. For example,some CTPs do not result in adequate protein expression. This can includeincorrect expression of the protein, with the production of proteins ofaltered size and incomplete activity in vivo. This can result inincomplete herbicide tolerance and complicate regulatory approval. Thepresent invention provides CTPs that, when used in combination with DMO,provide unexpected benefits including, but not necessarily limited to,improved levels of transport to the chloroplast, increased herbicidetolerance in DMO-expressing transgenic plants, desired levels of proteinexpression of the correct size, and appropriate post-translationalmodifications. One such example of a CTP providing unexpected benefitswhen in combination with DMO is the transit peptide CTP2, including thenucleic acids of SEQ ID NO:15 or 16, and including sequences encodingSEQ ID NOs:4 or 5. In other embodiments, a pea (Pisum sativum) Rubiscosmall subunit CTP coding sequence is used, such as represented by SEQ IDNO: 13 or encoding SEQ ID NO: 2. A DNA construct comprising a DMO codingsequence operably linked to a CTP2 and/or pea Rubisco small subunit CTPtransit peptide coding sequence thus forms one aspect of the invention,as does a protein encoded thereby.

Dicamba monooxygenase of Pseudomonas maltophilia strain DI6 (Herman etal., 2005; U.S. Patent Publication 20030115626; GenBank accessionAY786443, the DMO-encoding sequence of which is herein incorporated byreference) catalyzes the detoxification of the herbicide dicamba. DMO ispart of a 3-component system for detoxification of dicamba to thenon-toxic 3,6-dichlorosalicylic acid (3,6-DCSA), and as noted aboverequires reductase and ferredoxin functions for transfer of electrons.Since the endogenous plant ferredoxin that is involved in electrontransfer is localized in the plastids, in order to obtain efficientactivity of DMO and thus tolerance such as in dicots or improvedtolerance such as in monocots to dicamba, DMO is preferably targeted toplastids (e.g. chloroplasts).

Chloroplast transit peptides (CTPs) were tested for efficiency inallowing targeting and processing of DMO to plastids. Plastidlocalization and processing of the DMO in connection with these CTPsranged from none, or partial, to complete. Only some of the CTPs werefound to allow complete processing of DMO to a correct size. The abilityof any given CTP to provide for complete and efficient processing of DMOwas therefore unpredictable and surprising based on its protein ornucleotide sequences.

Further, it has also been found in Arabidopsis that without a properCTP, there is little or no expression of DMO correlating with little orno tolerance to dicamba. This suggests that chloroplast targeting isimportant for dicamba detoxification and hence tolerance. CTPs thatallow efficient processing of DMO will be useful in targeting DMO toplastids, such as chloroplasts, of crop plants thereby providing theadvantage of full DMO activity and higher tolerance to dicamba as wellas ease of product characterization and reduced cost of registration.

Chimeric DNA molecules comprising a DNA encoding a chloroplast transitpeptide operably linked to a DNA encoding dicamba monooxygenase can beprepared by molecular biological methods known to those skilled in thisart (e.g. Sambrook et al., 1989). CTPs operably linked to known DNAmolecules encoding DMO, including those identified in Table 1, areprovided by the invention for the improved expression of DMO in plants.

A chloroplast transit peptide from any gene that is encoded in thenucleus and the product of which targets a polypeptide to thechloroplast can be tested for efficient expression of DMO. Chloroplasttransit peptide sequences can be isolated or synthesized. The nucleotidesequence encoding a CTP may be optimized for expression in dicots,monocots, or both. The following transit peptides were tested byoperably linking each to a DMO coding sequence: PsRbcS-derived CTPs (SEQID NO:1 and 2: Pisum sativum Rubisco small subunit CTP; Coruzzi et al.,1984); AtRbcS CTP (SEQ ID NO:3: Arabidopsis thaliana Rubisco smallsubunit 1A CTP; CTP1; U.S. Pat. No. 5,728,925); AtShkG CTP (SEQ ID NO:4:Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS); CTP2; Klee et al., 1987); AtShkGZm CTP (SEQ ID NO:5:CTP2synthetic; codon optimized for monocot expression; SEQ ID NO:14 ofWO04009761); PhShkG CTP (SEQ ID NO:6: Petunia hybrida EPSPS; CTP4; codonoptimized for monocot expression; Gasser et al., 1988); TaWaxy CTP (SEQID NO:7: Triticum aestivum granule-bound starch synthase CTPsynthetic,codon optimized for corn expression: Clark et al., 1991): OsWaxy CTP(SEQ ID NO:8: Oryza sativa starch synthase CTP; Okagaki, 1992); NtRbcSCTP (SEQ ID NO: 9: Nicotiana tabacum ribulose 1,5-bisphosphatecarboxylase small subunit chloroplast transit peptide; Mazur, et al.,1985); ZmAS CTP (SEQ ID NO:10: Zea mays anthranilate synthase alpha 2subunit gene CTP; Gardiner et al., 2004); and RgAS CTP (SEQ ID NO:11:Ruta graveolens anthranilate synthase CTP; Bohlmann, et al., 1995). Thenucleotide sequences coding for SEQ ID NO:1-SEQ ID NO:11 are given inSEQ ID NO:12-SEQ ID NO:22, respectively.

Other transit peptides that may be useful include maize cab-m7 signalsequence (Becker et al., 1992; PCT WO 97/41228) and the pea (Pisumsativum) glutathione reductase signal sequence (Creissen et al., 1995;PCT WO 97/41228). CTPs with additional amino acids derived from thecoding region of the gene they are part of or are fused to, such asAtRbcS CTP which includes the transit peptide, 24 amino acids of themature Rubisco protein, and then a repeat of the last 6 amino acids ofthe transit peptide, can be utilized for producing DMO. ZmAS CTP alsocontain additional 18 amino acids derived from the coding region of thegene. Other CTPs with additional amino acids (for example 27 aminoacids) derived from the coding region of the gene they are part of, suchas PsRbcS CTP, followed by amino acids introduced by cloning methods(for example 3 amino acids) can also be utilized for producing DMO. CTPswith fewer amino acids (for example 21 amino acids) coding for a fulllength CTP such as RgAs CTP can also be utilized for producing DMO.Preferably, a nucleic acid sequence coding for a full length CTP isutilized. One or more nucleotide additions or deletions may be includedto facilitate cloning of a CTP. These additions or deletions may beafter or before other expression elements and coding regions, resultingin modification of one or more encoded amino acids, for instance at ornear a restriction enzyme recognition site.

In one embodiment, the invention relates to a nucleic acid sequenceencoding a chloroplast transit peptide that has at least 70% identity toa polypeptide sequence of any one or more of SEQ ID NOs: 1-11, includingat least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and greatersequence identity to these sequences, including 100% identity. Inparticular embodiments, the nucleic acid sequence encodes a chloroplasttransit peptide identical to one of SEQ ID NOs: 1-11. In anotherembodiment, the nucleic acid coding for the CTP has at least 70%sequence identity to a nucleic acid sequence of any one or more of SEQID NOs:12-22, including at least about 75%, 80%, 85%, 90%, 95%, 97%,98%, 99% and greater sequence identity, including 100% identity, to oneor more of these sequences. Polypeptide or polynucleotide comparisons ofthese and any other sequence as described herein may be carried out andidentity determined as is well known in the art, for example, usingMEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis.) with defaultparameters. Such software matches similar sequences by assigning degreesof similarity or identity.

DMO can be targeted to other organelles such as mitochondria by usingpre-sequences to make use of the ferrodoxin redox system present in thisorganelle. Alternatively, DMO can be targeted to both chloroplast andmitochondria by a dual-targeting peptide to make use of two ferrodoxinredox systems to work even more effectively. Such elements are known tothose skilled in the art. For example, mitochondrial pre-sequences aredescribed in Silva Filho et al., (1996). Nucleic acid sequences thatencode dual-targeting peptide sequences can be identified from thenucleic acids coding for the following proteins which are known betargeted to both chloroplasts and mitochondria: Zn-MP (Moberg et al.,2003), gluthathione reductase (Rudhe et al., 2002; Creissen et al.,1995) and histdyl-tRNA synthetase (Akashi et al., 1998). Examples ofDMO-encoding sequences that may be used in this regard are found, forexample, in the sequences encoding the polypeptides of SEQ ID NOs 24,26, 28, 30, 32, 34, 36, 38, 40, as shown in Table 1.

TABLE 1 DMO and DMO variants utilized. Pre- Pre- Predicted PRT DNAdicted dicted aa at DMO/or SEQ SEQ PRT aa at pos- aa at pos- positionCodon variant ID ID Length ition 2 ition 3 112 usage DMO- 24 23 340 AlaThr Cys dicot Cat(A) DMO- 26 25 340 Leu Thr Cys dicot Cat(L) DMO- 28 27340 Leu Thr Trp dicot Wat(L) DMO- 30 29 340 Ala Thr Cys bacteria Cnat(A)DMO- 32 31 340 Ala Thr Trp dicot Wat(A) DMO- 34 33 340 Thr PheTrp (at 111) bacterium Wnat(T) DMO- 36 35 340 Leu Thr Cys bacteriumCnat(L) DMO- 38 37 340 Leu Thr Trp monocot Wmc(L) DMO- 40 39 340 Ala ThrTrp monocot Wmc(A)

In some embodiments, the nucleic acid encoding a dicamba monooxygenasehas at least 70% identity to a sequence that encodes a polypeptide ofany of SEQ ID NOs:24, 26, 28, 30, 32, 34, 36, 38, or 40, including atleast about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and greater sequenceidentity to these sequences. In certain embodiments, the nucleic acidhas at least 70% sequence identity to a nucleic acid sequence of any ofSEQ ID NOs: 23, 25, 27, 29, 31, 33, 35, 37, or 39, including at leastabout 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, and greater sequenceidentity to one of these sequences. In further embodiments, a dicambamonooxygenase may be a variant of any such sequences and/or may be asynthetic DMO molecule engineered, for example, as described in U.S.Prov. Appl. Ser. No. 60/884,854, filed Jan. 12, 2007, entitled “DMOMethods And Compositions,” the entire disclosure of which isspecifically incorporated herein by reference.

Variants of DMOs having a capability to degrade auxin-like herbicides,as well as glyphosate or other herbicide tolerance genes can readily beprepared and assayed for activity according to standard methods. Suchsequences can also be identified by techniques known in the art, forexample, from suitable organisms including bacteria that degradeauxin-like herbicides, such as dicamba, or other herbicides (U.S. Pat.No. 5,445,962; Cork and Krueger, 1991; Cork and Khalil, 1995). One meansof isolating a DMO or other sequence is by nucleic acid hybridization,for example, to a library constructed from the source organism, or byRT-PCR using mRNA from the source organism and primers based on thedisclosed desaturases. The invention therefore encompasses use ofnucleic acids hybridizing under stringent conditions to a DMO encodingsequence described herein. One of skill in the art understands thatconditions may be rendered less stringent by increasing saltconcentration and decreasing temperature. Thus, hybridization conditionscan be readily manipulated, and thus will generally be a method ofchoice depending on the desired results. An example of high stringencyconditions is 5×SSC, 50% formamide and 42° C. By conducting a wash undersuch conditions, for example, for 10 minutes, those sequences nothybridizing to a particular target sequence under these conditions canbe removed.

Variants can also be chemically synthesized, for example, using theknown DMO polynucleotide sequences according to techniques well known inthe art. For instance, DNA sequences may be synthesized byphosphoroamidite chemistry in an automated DNA synthesizer. Chemicalsynthesis has a number of advantages. In particular, chemical synthesisis desirable because codons preferred by the host in which the DNAsequence will be expressed may be used to optimize expression. Not allof the codons need to be altered to obtain improved expression, butpreferably at least the codons rarely used in the host are changed tohost-preferred codons. High levels of expression can be obtained bychanging greater than about 50%, most preferably at least about 80%, ofthe codons to host-preferred codons. The codon preferences of many hostcells are known (e.g. PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP553494; and U.S. Pat. Nos. 5,689,052; 5,567,862; 5,567,600; 5,552,299and 5,017,692). The codon preferences of other host cells can be deducedby methods known in the art. Also, using chemical synthesis, thesequence of the DNA molecule or its encoded protein can be readilychanged to, for example, optimize expression (for example, eliminatemRNA secondary structures that interfere with transcription ortranslation), add unique restriction sites at convenient points, anddelete protease cleavage sites.

Modification and changes may be made to the polypeptide sequence of aprotein such as the DMO sequences provided herein while retaining ormodifying enzymatic activity as desired. Illustrative methods forgenerating DMO sequences are provided in U.S. Prov. Appl. Ser. No.60/884,854, filed Jan. 12, 2007. The following is a discussion basedupon changing the amino acids of a protein to create an equivalent, oreven an improved, modified polypeptide and corresponding codingsequences. It is known, for example, that certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures such asbinding sites on substrate molecules. Since it is the interactivecapacity and nature of a protein that defines that protein's biologicalfunctional activity, certain amino acid sequence substitutions can bemade in a protein sequence, and, of course, its underlying DNA codingsequence, and nevertheless obtain a protein with like properties. It isthus contemplated that various changes may be made in the DMO peptidesequences described herein or other herbicide tolerance polypeptides andcorresponding DNA coding sequences without appreciable loss of theirbiological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte et al., 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics (Kyte et al., 1982), theseare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is known in the art that amino acids may be substituted by otheramino acids having a similar hydropathic index or score and still resultin a protein with similar biological activity, i.e., still obtain abiological functionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent protein.In such changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred. Exemplary substitutions which take these and various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

A DNA construct comprising a CTP sequence operably linked to a DMOsequence can be expressed in test system such as protoplasts,transiently or stably transformed plant cells by operably linked them toa promoter functional in plants. Examples describing such promotersinclude U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No.5,641,876 (rice actin promoter; OsAct1), U.S. Pat. No. 6,426,446 (maizeRS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S.Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611(constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605,5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maizeL3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter aswell as a rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specificpromoter), U.S. Pat. No. 6,294,714 (light inducible promoters), U.S.Pat. No. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138(pathogen inducible promoters), U.S. Pat. No. 6,175,060 (phosphorusdeficiency inducible promoters), U.S. Pat. No. 6,388,170 (e.g. PC1SVpromoter), the PC1SV promoter of SEQ ID NO:41, U.S. Pat. No. 6,635,806(gamma-coixin promoter), and U.S. Pat. No. 7,151,204 (maize chloroplastaldolase promoter). Additional promoters that may find use are anopaline synthase (NOS) promoter (Ebert et al., 1987), the octopinesynthase (OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), theCaMV 35S promoter (Odell et al., 1985), the figwort mosaic virus35S-promoter (Walker et al., 1987), the sucrose synthase promoter (Yanget al., 1990), the R gene complex promoter (Chandler et al., 1989), andthe chlorophyll a/b binding protein gene promoter, etc. In the presentinvention, CaMV35S with enhancer sequences (e35S; U.S. Pat. Nos.5,322,938; 5,352,605; 5,359,142; and 5,530,196), FMV35S (U.S. Pat. Nos.6,051,753; 5,378,619), peanut chlorotic streak caulimovirus (PC1SV; U.S.Pat. No. 5,850,019), At.Act 7 (Accession # U27811), At.ANT1 (US PatentApplication 20060236420), FMV.35S-EF1a (US Patent Application20050022261), eIF4A10 (Accession # X79008) and AGRtu.nos (GenBankAccession V00087; Depicker et al, 1982; Bevan et al., 1983), ricecytosolic triose phosphate isomerase (OsTPI; U.S. Pat. No. 7,132,528),and rice actin 15 gene (OsAct15; U.S. Patent Application 2006-0162010)promoters may be of particular benefit.

A 5′ UTR that functions as a translation leader sequence is a DNAgenetic element located between the promoter sequence of a gene and thecoding sequence may be included between a promoter and CTP-DMO sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences include maize and petunia heat shock protein leaders (U.S.Pat. No. 5,362,865), plant virus coat protein leaders, plant rubiscoleaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No.5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), and AGRtunos(GenBank Accession V00087; Bevan et al., 1983) among others. (Turner andFoster, 1995). In the present invention, 5′ UTRs that may in particularfind benefit are GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No.5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), OsAct1 (U.S. Pat.No. 5,641,876), OsTPI (U.S. Pat. No. 7,132,528), OsAct15 (US PublicationNo. 20060162010), and AGRtunos (GenBank Accession V00087; Bevan et al.,1983).

The 3′ non-translated sequence, 3′ transcription termination region, orpoly adenylation region means a DNA molecule linked to and locateddownstream of a structural polynucleotide molecule and includespolynucleotides that provide polyadenylation signal and other regulatorysignals capable of affecting transcription, mRNA processing or geneexpression. The polyadenylation signal functions in plants to cause theaddition of polyadenylate nucleotides to the 3′ end of the mRNAprecursor. The polyadenylation sequence can be derived from the naturalgene, from a variety of plant genes, or from T-DNA genes. Thesesequences may be included downstream of a CTP-DMO sequence. An exampleof a 3′ transcription termination region is the nopaline synthase 3′region (nos 3′; Fraley et al., 1983). The use of different 3′nontranslated regions is exemplified (Ingelbrecht et al., 1989).Polyadenylation molecules from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9;Coruzzi et al., 1984), AGRtu.nos (Genbank Accession E01312), E6(Accession # U30508), and TaHsp17 (wheat low molecular weight heat shockprotein gene; Accession # X13431) in particular may be of benefit foruse with the invention.

In addition to expression elements described above, an intron may berequired in between a promoter and a 3′ UTR for expressing a codingregion, especially in monocots. An “intron” refers to a polynucleotidemolecule that may be isolated or identified from the interveningsequence of a genomic copy of a gene and may be defined generally as aregion spliced out during mRNA processing prior to translation.Alternately, introns may be synthetically produced. Introns maythemselves contain sub-elements such as cis-elements or enhancer domainsthat effect the transcription of operably linked genes. A “plant intron”is a native or non-native intron that is functional in plant cells. Aplant intron may be used as a regulatory element for modulatingexpression of an operably linked gene or genes. A polynucleotidemolecule sequence in a transformation construct may comprise introns.The introns may be heterologous with respect to the transcribablepolynucleotide molecule sequence. Examples of introns include the cornactin intron (U.S. Pat. No. 5,641,876), the corn HSP70 intron (ZmHSP70;U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412), and rice TPI intron(OsTPI; U.S. Pat. No. 7,132,528) and are of benefit in practicing thisinvention.

The CTP-DMO constructs can be tested for providing proper processing ofDMO in a test system such as protoplasts, or transiently or stablytransformed plant cells of monocots or dicots by methods known to thoseskilled in the art of plant tissue culture and transformation. Any ofthe techniques known in the art for introduction of transgene constructsinto plants may be used in accordance with the invention (see, forexample, Mild et al., 1993). Suitable methods for transformation ofplants are believed to include virtually any method by which DNA can beintroduced into a cell, such as by electroporation as illustrated inU.S. Pat. No. 5,384,253; microprojectile bombardment as illustrated inU.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861;and 6,403,865; Agrobacterium-mediated transformation as illustrated inU.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and6,384,301; and protoplast transformation as illustrated in U.S. Pat. No.5,508,184. Through the application of techniques such as these, thecells of virtually any plant species may be stably transformed, andthese cells may be developed into transgenic plants. Techniques that maybe particularly useful in the context of cotton transformation aredisclosed in U.S. Pat. Nos. 5,846,797, 5,159,135, 5,004,863, and6,624,344. Techniques for transforming Brassica plants in particular aredisclosed, for example, in U.S. Pat. No. 5,750,871; and techniques fortransforming soybean are disclosed in, for example, Zhang et al., 1999,U.S. Pat. No. 6,384,301, and U.S. Pat. No. 7,002,058. Techniques fortransforming corn are disclosed in WO9506722. Some non-limiting examplesof plants that may find use with the invention include alfalfa, barley,beans, beet, broccoli, cabbage, carrot, canola, cauliflower, celery,Chinese cabbage, corn, cotton, cucumber, dry bean, eggplant, fennel,garden beans, gourd, leek, lettuce, melon, oat, okra, onion, pea,pepper, pumpkin, peanut, potato, pumpkin, radish, rice, sorghum,soybean, spinach, squash, sweet corn, sugarbeet, sunflower, tomato,watermelon, and wheat.

After effecting delivery of exogenous DNA to recipient cells, the nextsteps in generating transgenic plants generally concern identifying thetransformed cells for further culturing and plant regeneration. In orderto improve the ability to identify transformants, one may desire toemploy a selectable or screenable marker gene with a transformationvector prepared in accordance with the invention. In this case, onewould then generally assay the potentially transformed cell populationby exposing the cells to a selective agent or agents, or one wouldscreen the cells for the desired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. Any suitable plant tissue culturemedia, for example, MS or N6 media (Murashige and Skoog, 1962; Chu etal., 1975); may be modified by including further substances such asgrowth regulators. Tissue may be maintained on a basic media with growthregulators until sufficient tissue is available to begin plantregeneration efforts, or following repeated rounds of manual selection,until the morphology of the tissue is suitable for regeneration,typically at least 2 weeks, then transferred to media conducive to shootformation. Cultures are transferred periodically until sufficient shootformation had occurred. Once shoot are formed, they are transferred tomedia conducive to root formation. Once sufficient roots are formed,plants can be transferred to soil for further growth and maturity.

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

Once a transgene has been introduced into a plant, that gene can beintroduced into any plant sexually compatible with the first plant bycrossing, without the need for ever directly transforming the secondplant. Therefore, as used herein the term “progeny” denotes theoffspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct prepared in accordance with the invention. A “transgenicplant” may thus be of any generation. “Crossing” a plant to provide aplant line having one or more added transgenes or alleles relative to astarting plant line, as disclosed herein, is defined as the techniquesthat result in a particular sequence being introduced into a plant lineby crossing a starting line with a donor plant line that comprises atransgene or allele of the invention. To achieve this one could, forexample, perform the following steps: (a) plant seeds of the first(starting line) and second (donor plant line that comprises a desiredtransgene or allele) parent plants; (b) grow the seeds of the first andsecond parent plants into plants that bear flowers; (c) pollinate aflower from the first parent plant with pollen from the second parentplant; and (d) harvest seeds produced on the parent plant bearing thefertilized flower.

The stably transformed plant tissues and plants can be tested forproviding dicamba tolerance by correct processing of DMO protein.Provision of dicamba tolerance in a crop plant can be used for designinga method for controlling weed growth in a growing environment comprisingapplying to the crop growing environment an amount of dicamba herbicideeffective to control weed growth. The dicamba herbicide is applied overthe top to the crop growing environment in an amount that does notdamage the crop plant or seed transformed with a CTP-DMO construct anddamages a crop plant of the same genotype lacking the CTP-DMO construct.

The preparation of herbicide compositions for use in connection with thecurrent invention will be apparent to those of skill in the art in viewof the disclosure. Such compositions, which are commercially available,will typically include, in addition to the active ingredient, componentssuch as surfactants, solid or liquid carriers, solvents and binders.Examples of surfactants that may be used for application to plantsinclude the alkali metal, alkaline earth metal or ammonium salts ofaromatic sulfonic acids, e.g., ligno-, phenol-, naphthalene- anddibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates,of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and offatty alcohol glycol ether sulfates, condensates of sulfonatednaphthalene and its derivatives with formaldehyde, condensates ofnaphthalene or of the naphthalenesulfonic acids with phenol andformaldehyde, condensates of phenol or phenolsulfonic acid withformaldehyde, condensates of phenol with formaldehyde and sodiumsulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-,octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylarylpolyether alcohols, isotridecyl alcohol, ethoxylated castor oil,ethoxylated triarylphenols, salts of phosphatedtriarylphenolethoxylates, lauryl alcohol polyglycol ether acetate,sorbitol esters, lignin-sulfite waste liquors or methylcellulose, ormixtures of these. Common practice in the case of surfactant use isabout 0.25% to 1.0% by weight, and more commonly about 0.25% to 0.5% byweight.

Compositions for application to plants may be solid or liquid. Wheresolid compositions are used, it may be desired to include one or morecarrier materials with the active compound. Examples of carriers includemineral earths such as silicas, silica gels, silicates, talc, kaolin,attaclay, limestone, chalk, loess, clay, dolomite, diatomaceous earth,calcium sulfate, magnesium sulfate, magnesium oxide, ground syntheticmaterials, fertilizers such as ammonium sulfate, ammonium phosphate,ammonium nitrate, thiourea and urea, products of vegetable origin suchas cereal meals, tree bark meal, wood meal and nutshell meal, cellulosepowders, attapulgites, montmorillonites, mica, vermiculites, syntheticsilicas and synthetic calcium silicates, or mixtures of these.

For liquid solutions, water-soluble compounds or salts may be included,such as sodium sulfate, potassium sulfate, sodium chloride, potassiumchloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride,ammonium acetate, ammonium formate, ammonium oxalate, ammoniumcarbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammoniumhydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodiumhydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammoniumcarbamate.

Other exemplary components in herbicidal compositions include binderssuch as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzedpolyvinyl acetate, carboxymethylcellulose, starch,vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, ormixtures of these; lubricants such as magnesium stearate, sodiumstearate, talc or polyethylene glycol, or mixtures of these; antifoamssuch as silicone emulsions, long-chain alcohols, phosphoric esters,acetylene diols, fatty acids or organofluorine compounds, and chelatingagents such as: salts of ethylenediaminetetraacetic acid (EDTA), saltsof trinitrilotriacetic acid or salts of polyphosphoric acids, ormixtures of these.

Dicamba may be used from about 2.5 g/ha to about 10,080 g/ha, includingabout 2.5 g/ha to about 5,040 g/ha, about 5 g/ha to about 2,020 g/ha,about 10 g/a to about 820 g/h and about 50 g/ha to about 1,000 g/ha,about 100 g/ha to about 800 g/ha and about 250 g/ha to about 800 g/ha.

The CTP-DMO constructs can be linked to one or more polynucleotidemolecules containing genetic elements for ascreenable/scorable/selectable marker and/or for a gene conferringanother desired trait. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase, and chloramphenicol acetyltransferase (Jefferson, 1987;Teeri et al., 1989; Koncz et al., 1987; De Block et al., 1984), greenfluorescent protein (GFP) (Chalfie et al., 1994; Haseloff and Amos,1995; and PCT application WO 97/41228). Non-limiting examples ofselectable marker genes are described in, e.g., Miki and McHugh, 2004.

The nucleotide molecule conferring another desired trait may include,but is not limited to, a gene that provides a desirable characteristicassociated with plant morphology, physiology, growth and development,yield, nutritional enhancement, disease or pest resistance, orenvironmental or chemical tolerance and may include genetic elementscomprising herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476;6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435;5,463,175), increased yield (U.S. Pat. Nos. RE38,446; 6,716,474;6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211;6,235,971; 6,222,098; 5,716,837), insect control (U.S. Pat. Nos.6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030;6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756;6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949;6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573;6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013;5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245; 5,763,241),fungal disease resistance (U.S. Pat. Nos. 6,653,280; 6,573,361;6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436;6,316,407; 6,506,962), virus resistance (U.S. Pat. Nos. 6,617,496;6,608,241; 6,015,940; 6,013,864; 5,850,023; 5,304,730), nematoderesistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S.Pat. No. 5,516,671), plant growth and development (U.S. Pat. Nos.6,723,897; 6,518,488), starch production (U.S. Pat. Nos. 6,538,181;6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production(U.S. Pat. Nos. 6,444,876; 6,426,447; 6,380,462), high oil production(U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; 6,476,295), modifiedfatty acid content (U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465;6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461;6,459,018), high protein production (U.S. Pat. No. 6,380,466), fruitripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition(U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; 6,171,640),biopolymers (U.S. Pat. Nos. RE37,543; 6,228,623; 5,958,745 and U.S.Patent Publication No. US20030028917), environmental stress resistance(U.S. Pat. No. 6,072,103), pharmaceutical peptides and secretablepeptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075; 6,080,560),improved processing traits (U.S. Pat. No. 6,476,295), improveddigestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No.6,166,292), industrial enzyme production (U.S. Pat. No. 5,543,576),improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat.No. 5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), fiberproduction (U.S. Pat. Nos. 6,576,818; 6,271,443; 5,981,834; 5,869,720)and biofuel production (U.S. Pat. No. 5,998,700). Any of these or othergenetic elements, methods, and transgenes may be used with the inventionas will be appreciated by those of skill in the art in view of theinstant disclosure.

Alternatively, the one or more polynucleotide molecule linked to CTP-DMOconstruct can effect the above mentioned plant characteristic orphenotype by encoding a RNA molecule that causes the targeted inhibitionof expression of an endogenous gene, for example, via antisense,inhibitory RNA (RNAi), or cosuppression-mediated mechanisms. The RNAcould also be a catalytic RNA molecule (i.e., a ribozyme) engineered tocleave a desired endogenous mRNA product. Thus, any polynucleotidemolecule that encodes a transcribed RNA molecule that affects aphenotype or morphology change of interest may be useful for thepractice of the present invention.

The present invention also discloses a method for producing food, feed,or an industrial product comprising a plant containing a CTP-DMOconstruct or a part of such a plant and preparing the food, feed, fiber,or industrial product from the plant or part thereof, wherein the foodor feed is grain, meal, oil, starch, flour, or protein and theindustrial product is biofuel, fiber, industrial chemicals, apharmaceutical, or nutraceutical.

Another aspect of the invention relates to a method for improving thestandability of a monocot plant comprising: a) obtaining and growing aplant produced by crossing a parent plant with itself or with a secondplant, wherein the parent plant and/or the second plant comprises theDNA construct and the dicamba tolerant plant inherits the DNA constructfrom said parent plant and/or the second plant; and b) treating theplant with dicamba. Parameters relating to standability may be measured,for instance including brace root number, shape, length or structure;percent lodging; and yield. In certain embodiments, the plant is a cornplant.

EXAMPLES

The following examples are included to illustrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Preparation of CTP-DMO Constructs for Transformation

DNA constructs as shown in Table 2 were prepared according to standardmethods (e.g. Sambrook et al., 1989), comprising a CTP operably linkedwith a DMO gene, or a variant thereof, between a plant promoter and apolyadenylation signal sequence. These constructs were tested in eithera corn protoplast system or in stably transformed Arabidopsis or soybeanplants as described below.

TABLE 2 Processing of DMO and DMO variants by different CTPs. % totalexpression PRT DNA # Band Size (band SEQ SEQ DMO of (band 1/band 1/bandpMON Promoter CTP ID ID version 3′UTR Test System Bands 2) 2) 84254PCISV PsRbcS CTP with coding 1 12 DMOc- RbcS Soybean Leaf 2 41 kDa/38kDa 10/90 region native(A) 2-E9 58498 PCISV PsRbcS CTP with coding 1 12DMOc- RbcS Soybean Leaf 2 41 kDa/38 kDa 50/50 region native(A) 2-E973749 PCISV PsRbcS CTP with coding 1 12 DMOc-dc(A) E6 Arabidopsis 2 41kDa/38 kDa 50/50 region Leaf 73725 PCISV PsRbcS CTP without coding 2 13DMOw- Nos Arabidopsis 1  38 kDa 100 region dc(A) Leaf 73728 PCISV PsRbcSCTP without coding 2 13 DMOw-dc(L) Nos Arabidopsis 1  38 kDa 100 regionLeaf 73729 PCISV AtRbcS CTP (CTP1) 3 14 DMOw- Nos Corn 1 >41 kDa 100dc(A) Protoplasts 73708 PCISV AtRbcS CTP (CTP1) 3 14 DMOw- Hsp17 Corn1 >41 kDa 100 mc(L) Protoplasts 73698 FMV AtRbcS CTP (CTP1) 3 14DMOc-dc(L) RbcS Arabidopsis 1 >41 kDa 100 35S 2-E9 Leaf 73731 CaMVAtShkG CTP (CTP2) 4 15 DMOw- Hsp17 Corn 1 ~38 kDa 100 35S- mc(L)Protoplasts enh 73740 PCISV AtShkG CTP (CTP2) 4 15 DMOc- Nos Corn 1 ~38kDa 100 native(L) Protoplasts 73713 PCISV AtShkG CTP 5 16 DMOw- Hsp17Corn 1 ~38 kDa 100 (CTP2synthetic) mc(L) Protoplasts 73742 PCISV AtShkGCTP 5 16 DMOc- Hsp17 Corn 1 ~38 kDa 100 (CTP2synthetic) native(L)Protoplasts 73724 PCISV AtShkG CTP 5 16 DMOw- Nos Arabidopsis 1 ~38 kDa100 (CTP2synthetic) dc(A) Leaf 73727 PCISV AtShkG CTP 5 16 DMOw-dc(L)Nos Arabidopsis 2 >38 kDa/ 50/50 (CTP2synthetic) Leaf ~38 kDa 73736 CaMVPhShkG CTP 6 17 DMOw- Hsp17 Corn 1 ~38 kDa 100 35S- (CTP4synthetic)mc(L) Protoplasts enh 73747 PCISV PhShkG CTP 6 17 DMOw- Hsp17 Corn 1 ~38kDa 100 (CTP4synthetic) mc(L) Protoplasts 73714 PCISV TaWaxyCTPsynthetic 7 18 DMOw- Hsp17 Corn 0 — — mc(L) Protoplasts 73716 PCISVTaWaxy CTPsynthetic 7 18 DMOw- Hsp17 Corn 0 — — mc(L) Protoplasts 73733CaMV OsWaxy CTP 8 19 DMOw- Hsp17 Corn 1 <38 kDa 100 35S- mc(L)Protoplasts enh 73734 CaMV NtRbcS CTP 9 20 DMOw- Hsp17 Corn 2 >41 kDa/38kDa 75/25 35S- mc(L) Protoplasts enh 73732 CaMV ZmAS CTP 10 21 DMOw-Hsp17 Corn 1 >38 kDa 100 35S- mc(L) Protoplasts enh 73735 CaMV RgAS CTP11 22 DMOw- Hsp17 Corn 1 >38 kDa 100 35S- mc(L) Protoplasts enh

Example 2 Analysis of CTP-DMO Constructs in Corn Protoplasts

Corn (maize) leaf mesophyll protoplasts were prepared from 12 days oldetiolated seedlings (from a LH200×LH5 cross). The middle parts of thesecond leaves (about 6 cm in length) were cut to 0.5-mm strips with arazor blade and digested in a flask in an enzyme solution containing 2%(w/v) cellulase RS, 0.3% (w/v) macerozyme R10 (both from Karlan ResearchProducts Corp, Santa Rosa, Calif.), 0.6 M mannitol, 10 mM MES (pH 5.7)and 1 mM CaCl₂, for no more than 2 hr at 23° C. after 30 minutes ofvacuum infiltration. Protoplasts from infiltrated and digested leaftissue were released by shaking the flask by hand for 5 min andseparated by filtering through a 60-μm nylon mesh. The protoplasts werecollected by centrifugation at 150 g for 2 min, washed in cold 0.6 Mmannitol solution once, centrifuged, and resuspended at a 2×10⁶/mL incold 0.6 M mannitol. The protoplasts were then transformed with 12.5 μgDNA using polyethylene glycol (PEG) and incubated at room temperaturefor 16 to 20 hr.

The protoplasts were stored at −80° C. until analysis by western blot.The protoplasts were thawed on ice and 1-3 volumes of 2× Laemmli samplebuffer/dye (BioRad) with 5.0% β-ME was added to the protoplasts.Aliquots of the protoplast protein samples were then heated to about100° C. for 5 minutes and loaded onto a pre-cast Tris-HCL 10%polyacrylamide gel. Electrophoresis was performed at a constant currentof about 80-100 Amps for about 35 minutes. Protein from the gel waselectro-transferred to a 0.2 micron nitrocellulose membrane for 1-3hours at a constant voltage of 100 V. The membrane was blocked for onehour at room temperature or overnight at 4° C. with 5% (w/v) dry milk inTBST. The membrane was probed with a 1:200 dilution of goat anti-DMOantibody in TBST for one hour. Excess antibody was removed using three 5min washes with TBS. The membrane was probed with peroxidase-conjugatedrabbit anti-goat IgG (Sigma, St. Louis, Mo.) at a dilution of 1:7,500 in0.5% (w/v) dry milk in TBST for one hour. Excess peroxidase-conjugatewas removed using three 5 min washes with TBST. All procedures,including blocking, and all other incubations were performed at roomtemperature, except where noted. Immunoreactive bands were visualizedusing the ECL detection system (Amersham Biosciences, Piscataway, N.J.)and exposed to Kodak BioMax™ MS film. The presence of immunoreactivebands of the appropriate size indicates proper processing andlocalization of DMO (Table 1). Thus, for instance, use of CTP4 operablylinked to DMO and transformed into corn protoplasts results in a 38 kDaimmunoreactive band following western blot analysis.

Example 3 Testing of Various CTP-DMO Constructs in Arabidopsis

Arabidopsis thaliana ecotype Columbia plants were transformed accordingto the method developed by Clough and Bent (1998). Seeds obtainedthrough this method were plated on a plant culture selection mediumcontaining dicamba at various concentrations from 0.5, 1.0, to 2.0 or4.0 mg/liter. The plates were incubated for 48 hours at 4° C. and thentransferred to a Percival incubator set at 23.5° C. with a photoperiodof 16 hours. Seeds that were transformed with CTP-DMO constructs grewinto plants on dicamba containing medium and developed primary andsecondary leaves, while the untransformed seed and negative segregantseither died or did not develop primary and secondary leaves. Thetransgenic plants that tested positive for the 3′ UTR by Invader® PCRassay were used further for analysis.

Three to five leaf punches from the transgenic Arabidopsis plants wereused for western blot analysis. Protein extraction was performed with500-1000 μl PSBT and 4 glass beads in a Harbil paint shaker for 3minutes. Samples were spun at 3000 rpm for 3 minutes at 4° C. An equalvolume of 2× Laemmli sample buffer/dye (cat. No. 161-0737 BioRad) with5.0% β-ME was added to aliquots of the supernatant. Remaining steps ofthe western blot analysis were the same as in Example 2. The presence ofimmunoreactive bands of the appropriate size indicates proper processingand localization of DMO (Table 2). For instance, as shown in Table 2, ina comparison of bands seen following transformation of Arabidopsis withpMON73749 or pMON73725, use of RbcSnoc-CTP, lacking the 27 aa codingsequence derived from pea Rubisco enzyme resulted in properly processedDMO localized to the chloroplast, while use of the RbcS CTP includingthe 27 aa coding sequence resulted in two immunoreactive bands.

Example 4 Testing of CTP-DMO Constructs in Soybean

Transgenic soybean (e.g. cvs. Thorne, NE3001 and A3525) plants wereobtained by Agrobacterium-mediated transformation of soybean usingstandard procedures (e.g. Zhang et al., 1999; U.S. Pat. No. 7,002,058).Three to five leaf punches from the transgenic soybean plants were usedfor western blot analysis. Protein extraction was performed with500-1000 μl PSBT and 4 glass beads in a Harbil paint shaker for 3 min.Samples were spun at 3000 rpm for 3 minutes at 4° C. An equal volume of2× Laemmli sample buffer/dye (BioRad) w/5.0% β-ME was added to aliquotsof the supernatant. The remaining steps of the western blot analysiswere the same as in Example 2. The presence of immunoreactive bands ofthe appropriate size indicates proper processing and localization of DMO(Table 2).

Soybean plants that were transformed with a construct coding for a DMOlinked to a pea Rubisco transit peptide attached to an additional 24amino acids of the Rubisco coding region, and 3 amino acids due tointroduction of restriction enzyme recognition sites, showed an injuryrate of 17-20% when treated with 0.5 lb of dicamba at pre emergencestage followed by 2 lb of dicamba at post emergence (V6) stage. Thiscompares with soybean plants that were transformed with a constructcoding for a DMO linked to a pea Rubisco transit peptide only, thatshowed an injury rate of about 12%. These results indicate that use of atransit peptide without additional amino acids results in production ofa single DMO activity (rather than multiple partially or differentlyprocessed polypeptides) and higher tolerance to dicamba. Production of asingle form of the enzyme will also lead to ease of productcharacterization and reduced cost of registration.

Example 5 Efficient Production of DMO and Higher Tolerance to DicambaRequires a CTP

Arabidopsis thaliana ecotype Columbia plants were transformed withseveral constructs (FIG. 1) as described in Example 3. Transformed seedswere selected on a plant tissue culture medium containing dicamba atvarious concentrations from 0.5, 1.0, to 2.0 mg/liter. Seeds that weretransformed with CTP-DMO constructs grew into plants on dicambacontaining medium and developed primary and secondary leaves, while theuntransformed seed and negative segregants either died or did notdevelop primary and secondary leaves. The transgenic plants that grewand tested positive for the DMO gene were used further for analysis.

As shown in FIG. 1, plants that were transformed with constructs withouta CTP exhibited little or no tolerance to dicamba. Soybean plantstransformed with a DNA construct coding for a DMO without linking it toa CTP showed no pre emergence tolerance whereas plants transformed withconstructs where the DMO was linked to the CTP showed both pre and postemergence tolerance to dicamba when treated with 0.5 lb/a of dicamba atpre emergence stage followed by 2 lb/a of dicamba at post emergence (V6)stage.

Example 6 Production of Dicamba Tolerant Transgenic Corn Plants

To test the use of a DMO gene in providing dicamba tolerance tomonocots, transgenic corn plants were produced that comprise a DMO gene(e.g. SEQ ID NOS: 29, 33, 35, 37, 39) with or without a transit peptide(e.g. TaWaxy, CTP1, CTP2synthetic, CTP4) under the control of plant geneexpression elements such as a promoter (e.g. PC1SV, e35S, OsAct1, OsTPI,OsAct15), and an intron (e.g. OsAct1, OsAct15, OsTPI, ZmHSP70). Thisexpression element contains first intron and flanking UTR exon sequencesfrom the rice actin 1 gene and includes 12 nt of exon 1 at the 5′ endand 7 nt of exon 2 at the 3′ end), and a 3′UTR (e.g. TaHsp17).

Transgenic corn plants were produced essentially by the method describedin U.S. patent application 20040244075. Transgenic corn events havingsingle copy were evaluated for dicamba tolerance at a single locationreplicated trial. Six events from each of the six constructs were used.The experimental design was as follows: rows/entry: 1; treatment: 0.5lb/a of dicamba at V3 stage followed by 1 lb/a of dicamba at V8 stage(Clarity®, BASF, Raleigh, N.C.); replications: 2; row spacing: 30inches; plot length: minimum 20 feet; plant density: about 30plants/17.5 ft.; alleys: 2.5 feet. The entire plot was fertilizeduniformly to obtain an agronomically acceptable crop. A soil insecticidesuch as Force® 3G (Syngenta Crop Protection, Greensboro, N.C., USA) at 5oz. per 1000 ft. of row for control of corn rootworm was applied atplanting time. If black cutworm infestation was observed, POUNCE® 3.2ECat 4 to 8 oz. per acre rate (FMC Corporation, Philadelphia, Pa.) wasused. In addition, an insecticide spray program was used to control allabove ground lepidopteran pests including European corn borer, cornearworm, and fall armyworm. POUNCE® 3.2EC at 4 to 8 oz. per acre wasapplied every 3 weeks to control lepidopteran pests; about 4applications were made. The plot was kept weed free with a pre-emergenceapplication of a herbicide such as Harness® Xtra 5.6L (Monsanto, St.Louis, Mo.) and Degree Xtra® (Monsanto, St. Louis, Mo.). If weed escapeswere observed in the untreated check, they were controlled by handweeding or a post-emergence application of PERMIT (Monsanto, St. Louis,Mo.) or BUCTRIL® (Bayer, Research Triangle Park, N.C.) over the entiretrial.

Corn inbred lines transformed with DNA constructs comprising a DMOtransgene were tested for dicamba tolerance by measuring brace rootinjury when treated with 0.5 lb/a of dicamba at V3 stage followed by 1lb/a of dicamba at V8 stage. Brace root injury was evaluated visually bycounting the number of plants in a row showing an “atypical” morphologyof having the brace roots fused as compared to a typical morphology of“finger-like” structure. As shown in Table 3, corn plants transformedwith DNA constructs coding for a DMO without linking it to a CTP(pMON73699, pMON73704) showed higher level of brace root injury, i.e.lower level of protection upon dicamba treatment. The constructs codingfor a DMO linked to a CTP (pMON73716, pMON73700, pMON73715, pMON73703)showed lower level of brace root injury, i.e. higher level of protectionupon dicamba treatment.

TABLE 3 Percentage brace root injury exhibited by transgenic corn plantstransformed with DNA constructs carrying DMO when tested for dicambatolerance. Brace Inbreds/ root Constructs Details injury 01CSI6Susceptible inbred to dicamba 95.4 LH244 Resistant inbred to dicamba93.8 pMON73699 PC1SV/I-OsAct1/DMO-Wmc/TaHsp17 93.2 pMON73704e35S/I-OsAct1/DMO-Wmc/TaHsp17 91.3 pMON73716PC1SV/I-OsAct1/TaWaxy/DMO-Wmc/TaHsp17 78.8 pMON73700PC1SV/I-OsAct1/CTP1/DMO-Wmc/TaHsp17 74.4 pMON73715PC1SV/I-OsAct1/CTP2syn/DMO-Wmc/TaHsp17 68.2 pMON73703e35S/I-OsAct1/CTP1/DMO-Wmc/TaHsp17 68.8

From these studies in diverse plant species (also, e.g. Examples 3, 4and 8), a chloroplast transit peptide is useful for efficient targetingof DMO and full production of DMO activity, leading to higher toleranceto dicamba. Further, expression of a CTP-DMO provides pre-emergencetolerance to dicamba in corn.

Example 7 Construction of Efficient DMO Expression Units

Several genetic elements can influence efficient expression of a genesuch as a promoter, chloroplast transit peptide sequence, an intron,5′UTR, coding region of the gene, 3′UTR. However, it is not obviouswhich combination will work the best. Efficient DMO expression units orconstructs are required to produce improved products such as a dicambatolerant seed and plant. Several DMO expression units were constructedby operably linking one of each various promoters, CTPs, DMO variants,and 3′UTRs to obtain efficient DMO expression units for productdevelopment. These constructs were transformed into soybean by methodsknown in the art (e.g. U.S. Pat. No. 6,384,301, U.S. Pat. No. 7,002,058or Zhang et al., 1999). Transgenic seeds were obtained and tested forpre- and post-emergence tolerance to dicamba herbicide. Table 4 showsthe % injury caused by dicamba (lower injury means higher tolerance)when seeds and plants were treated with 0.5 lb/acre of dicambapre-emergent followed by 2 lb/acre of dicamba post-emergent at V6 stage.Seeds transformed with DNA constructs pMON68939 and pMON73723 thatcarried no CTP were unable to tolerate pre-emergent application ofdicamba indicating that targeting of DMO to chloroplast is required toobtain pre-emergence tolerance to dicamba. Plants transformed withpMON68939 and pMON73723 (without CTP) that were treated with dicamba atpost-V3 stage at 1 lb/a rate showed injury rate of 55% and 57%respectively similar to the wild type soybean (60%) whereas the plantstransformed with pMON68938 (with CTP) showed very little injury. Theseresults indicate that a CTP is required for obtaining both pre and postemergence tolerance to dicamba in soybean.

TABLE 4 Percentage injury exhibited by soybean plants transformed with aspecific DMO expression unit and treated with dicamba pre-emergent andpost-emergent. pMON Expression Unit designation % InjuryPC1SV/CTP2syn/DMO-Wat(A)/nos 73724  9 e35S/CTP1/DMO-Wat(L)/nos 68938 12PC1SV/RbcSnoc/DMO-Wat(A)/nos 73725 12 PC1SV/RbcSnoc/DMO-Wat(L)/nos 7372812 PCSV/CTP1/DMO-Wat(A)/nos 73729 13 PC1SV/CTP2syn/DMO-Wat(L)/nos 7372713 ANT1/CTP1/DMO-Wat(L)/nos 68945 14 PC1SV/RbcSnoc/DMO-Wat(A)/nos 7373015 PC1SV/RbcS-CTP/DMO-Cnat(A)/nos 68934 17 Act7/CTP1/DMO-Wat(L)/nos68942 17 FMV.35S-EF1a/CTP1/DMO-Wat(L)/nos 68940 17PC1SV/RbcS-CTP/DMO-Cnat(A)/E9 84254 20 FMV/CTP1/DMO-Wat(L)/nos 68941 29eIF4A10/CTP1/DMO-Wat(L)/nos 68943 60 e35S/CTP1/DMO-Cat(A)/nos 68937 62e35S/CTP1/DMO-Cnat(L)/nos 68946 73 e35S/DMO-Wat(A)/nos 68939 100 (Pre)PC1SV/DMO-Wat(A)/nos 73723 100 (Pre)

Example 8 Production of Dicamba Tolerant Transgenic Cotton Plants

To test the use of DMO gene in providing dicamba tolerance to cotton,transgenic cotton plants were produced. Several DNA constructs carryinga DMO coding region (e.g. SEQ ID NOS: 23, 25, 27, 29, 31, 35) with atransit peptide (e.g., PsRbcS CTP, CTP1, CTP2) under the control ofplant gene expression elements such as a promoter (e.g. PC1SV, FMV, ore35S), and a 3′UTR (e.g. E6; Accession #U30508) were produced andtransformed into cotton (Gossypium hirsutum) as follows. Media used arenoted in Table 5.

Seedlings of cotton cv Coker 130 were grown in vitro and hypocotylsections were cut and inoculated with a liquid suspension ofAgrobacterium tumefaciens carrying a DNA construct, blot dried, andco-cultured for 2 days. Inoculated hypocotyl explants were thentransferred to glucose selection medium for 4 weeks, sucrose selectionmedium for 1 week, and to glucose selection medium for an additional 4weeks for inducing callus. The cultures were incubated in 16/8(light/dark) cycle and 28° C. temperature. Kanamycin resistant calliwere then transferred to UMO medium and cultured in the dark for 16-24weeks at 28-30° C. for inducing embryogenic callus. The embryogeniccallus was then harvested from these calli and maintained for up to 4-16weeks in the dark at 28-30° C. on TRP+ medium. Small embryos from theembryogenic callus were harvested and germinated on SHSU medium in 16/8(light/dark) cycle and 28-30° C. temperature. Plantlets that appearednormal were then transferred to soil to obtain mature cotton plants. Thetransgenic nature of transformants was confirmed by DNA testing.

TABLE 5 Composition of various media used for cotton transformation.Amount/L Components Glucose Sucrose UMO TRP+ SHSU MS basal salts(Phytotech.) 4.33 g 4.33 g 4.33 g 4.33 g — Gamborg's B5 vitamins(Phytotech) (500X) 2 ml 2 ml 2 ml 2 ml — 2,4-D (1 mg/ml) 0.1 ml 0.1 ml —— Stewart and Hsu majors (10X) — — — — 100 ml Stewart and Hsu minors(100X) — — — — 10 ml Steward and Hsu organic (100X) — — — — 10 mlKinetin (0.5 mg/ml) 1 ml 1 ml — — — Chelated iron (100X) — — — — 1.5 mlGlucose 30 g 30 g 30 g 30 g 5 g Potassium nitrate — — — 1.9 g — Caseinhydrolysate — — — 0.1 g — pH 5.8 5.8 5.8 5.8 6.8 Phytagel (Sigma) 2.5 g2.5 g — — — Gelrite (Kelco) — — 3.5 g 3.5 g 2.2 g Carbenicillin (250mg/ml) 1.7 ml 1.7 ml 1.7 ml 1.7 ml — Cefotaxime (100 mg/ml) 1 ml 1 ml 1ml 1 ml — Benlate (50 mg/ml) — — — 1 ml 1 ml Kanamycin (50 mg/ml)0.8-1.0 ml 0.8-1.0 ml 1 ml — — Sucrose — 0.1 g — — — Ascorbic acid — —100 mg

Transformed cotton plants that comprise such a DNA construct, eachcomprising a different combination of a DMO coding region with a transitpeptide, a promoter, and a 3′UTR, were treated with dicamba (Clarity®,BASF, Raleigh, N.C.) as a post-emergent treatment at V4-5 growth stageat the rate of 561 g ae/ha (0.5 lb/a) and found to be tolerant whereasuntransformed cotton plants showed an injury rate of 79% to 86%.Transgenic plants showing more than 95% tolerance (equal to less than 5%injury) were selected for further studies. Transgenic plants were alsotolerant to a subsequent post-emergent treatment of dicamba. Forexample, the plants that were treated with 0.5 lb/acre of dicamba atV3-4 stage followed by either 1 or 2 lb/acre of dicamba at V5 or laterstages were still tolerant to dicamba. This examples shows that a DMOgene can provide dicamba tolerance to cotton at various stages of growththus enabling application of dicamba at various stages to obtaineffective weed control.

Example 9 Method for Improving Standability of Corn

Certain monocots such as corn produce brace roots which grow from thenodes above the soil surface and help support the plant and scavenge theupper soil layers for water and nutrients during the reproductivestages. A healthy brace root system becomes important if the plants aresubjected to high winds or when the underground root system becomesweaker by root worm infection or under soil water deficit. Syntheticherbicide such as dicamba and 2,4-D are permitted for use on monocotssuch as corn for broad leaf weed control. For post-emergent weed controlin corn, dicamba is the 5th most widely used herbicide. Although theoptimal rate for broad leaf weed control is between 280 to 560grams/hectare (g/h) or 0.25 to 0.5 lb/acre, the average use rate in cornis 168 g/h or 0.15 lb/acre as at higher rates and under certainenvironmental conditions such as on hot days, dicamba can injure corn.In addition, several corn hybrids such as DKC61-42, DKC64-77, DKC63-46,DKC66-21 and DKC61-44 and inbreds such as 01CSI6, 16IUL2, 70LDL5, and90LCL6 are sensitive to dicamba applications. The sensitivity ismanifested in many ways such as occurrence of onion leafing, tasselmalformation, reduced plant height, or abnormal brace root formatione.g. fused or twisted root formation. The brace roots become gnarled,tending to grow together and not growing into the soil to support theplant. This may result in poor standability of a corn crop, highersusceptibility to lodging, and eventually yield loss. Several herbicideproducts that contain dicamba, for example Clarity®, BANVEL, MARKSMAN,DISTINCT, NORTHSTAR, and CELEBRITY PLUS, can cause these effects.Increasing tolerance of corn to dicamba will also be useful inprotecting corn fields planted closer to crop species such as soybeanand cotton that are tolerant to dicamba and where a higher rate ofdicamba application is permitted.

The present example provides a method for improving standability of cornand other monocots by incorporating a DMO gene in corn and treating cornwith dicamba. In one embodiment, the DMO gene is expressed under thecontrol of a constitutive promoter that is also capable of expressingDMO in nodal region and/or in brace roots. In another embodiment the DMOgene is expressed under the control of a chimeric constitutive andnode/brace root specific promoter. In another embodiment the DMO gene isexpressed under the control of a root specific promoter such as RCc3 ora variant thereof (e.g. SEQ ID NOs:1-6 as found in US20060101541). Theexpression of DMO in brace roots results in no or less injury to braceroots resulting in better standability of corn, less lodging, andtherefore better yield.

R1 or F1 seeds of three single copy events from corn plants transformedwith various DMO constructs (outlined in Table 6) were germinated in4.0″ trays. Healthy plants were transplanted into about 10″ pots.Germination and growth media comprised of Redi-earth™ (Scotts-SierraHorticultural Products Co., Marysville, Ohio). The pots were placed oncapillary matting in 35 inch×60 inch fiberglass watering trays forsub-irrigation for the duration of the test period so as to maintainoptimum soil moisture for plant growth. The pots were fertilized withOsmocote (14-14-14 slow release; Scotts-Sierra Horticultural ProductsCo., Marysville, Ohio) at the rate of 100 gm/cu. ft. to sustain plantgrowth for the duration of greenhouse trials. The plants were grown ingreenhouses at 29°/21° C. day/night temperature, with relative humiditybetween 25%-75% to simulate warm season growing conditions of latespring. A 14 h minimum photoperiod was provided with supplemental lightat about 600 μE as needed.

Dicamba applications were made with the track sprayer using a Teejet9501E flat fan nozzle (Spraying Systems Co, Wheaton, Ill.) with airpressure set at a minimum of 24 psi (165 kpa). The spray nozzle was keptat a height of about 16 inches above the top of plant material forspraying. The spray volume was 10 gallons per acre or 93 liters perhectare. Applications were made when plants had reached V 4-5 leafstage.

Plants of a corn inbred line transformed with DNA constructs comprisinga DMO expression unit were tested for brace root injury and lodging bytreating with 2 lb/acre or 4 lb/acre of dicamba at V4-5 stage andevaluating the plants for brace root injury (0%; no visible plantinjury) to 100% (complete death of plant); and lodging (degree ofleaning) at 24 DAT.

As shown in Table 6, corn plants transformed with the DNA constructshaving a DMO expression unit showed no or little brace root injury andlodging as compared to untransformed control inbred line and plantstransformed with a selectable marker expression unit only (pMON73746).This example shows that DMO containing plants provide can used toprovide improved standability when treated with dicamba.

TABLE 6 Corn plants transformed with various DMO constructs show no orlittle injury to brace roots and lodging when treated with dicamba.Dicamba Application Level 2 lb/acre 4 lb/acre % brace root injury andInbred/ lodging at construct Event Details of construct  24 DAT  24 DATcontrol 17.5 31.7 73746 R1 S214540 No DMO expression unit 20.0 28.173746 F1 S215886 33.8 40.0 73703 F2 S183001 e35S/I-OsAct1/CTP1- 0.9 1.0DMO Wmc/ TaHsp17 73744 F1 S208388 OsAct1/I-OsAct1/ 0.0 0.2CTP2syn-DMOWmc/ TaHsp17 73744 F1 S208373 0.0 0.4 73744 F1 S208382 0.00.0 73747 R1 S207612 PClSV/I-OsAct1/ 0.6 1.8 CTP4-DMOWmc/ TaHsp17 73747R1 S207608 0.8 1.0 73747 R1 S208367 0.0 0.0 73743 R1 S208476PCSV/I-OsAct1/ 3.9 19.6 CTP2syn/DMO-Cmc/ TaHsp17 73743 R1 S208469 1.322.9 73743 R1 S208071 2.7 13.5 73742 R1 S213404 PClSV/I-OsAct1/ 0.0 0.0CTP2syn-DMO-Cnat/ TaHsp17 73742 R1 S213395 0.2 0.4 73742 R1 S212111 0.40.5

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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The invention claimed is:
 1. A recombinant DNA molecule comprising a DNAsequence encoding a chloroplast transit peptide operably linked to a DNAsequence encoding dicamba monooxygenase, wherein the DNA sequenceencoding the chloroplast transit peptide encodes SEQ ID NO:4.
 2. Therecombinant DNA molecule of claim 1, wherein the DNA sequence encodingthe chloroplast transit peptide comprises SEQ ID NO:15.
 3. Therecombinant DNA molecule of claim 1, wherein the DNA sequence encodingdicamba monooxygenase encodes a polypeptide selected from the groupconsisting of SEQ ID NOs: 26, 28, 32, 34, 36, 38, and
 40. 4. Therecombinant DNA molecule of claim 3, wherein the DNA sequence isselected from the group consisting of SEQ ID NOs: 25, 27, 31, 33, 35,37, and
 39. 5. A DNA construct comprising the DNA molecule of claim 1operably linked to a promoter.
 6. The DNA construct of claim 5, whereinthe promoter is selected from the group consisting of a PC1SV promoter,a FMV35S promoter, an At.ANT1 promoter, an FMV.35S-EF1a promoter, aneIF4A10 promoter, an AGRtu.nos promoter, a rice cytosolic triosephosphate isomerase (OsTPI) promoter, a rice actin 15 gene (OsAct15)promoter, and a gamma coixin promoter.
 7. The construct of claim 5,wherein the promoter is functional in a plant cell.
 8. A plant celltransformed with the DNA construct of claim
 5. 9. The cell of claim 8,wherein the plant cell is a dicotyledonous plant cell.
 10. The cell ofclaim 8, wherein the plant cell is a monocotyledonous plant cell. 11.The cell of claim 8, wherein the plant cell is a soybean, cotton, maize,or rapeseed plant cell.
 12. A plant tissue culture comprising the cellof claim
 8. 13. The plant tissue culture of claim 12, comprising adicotyledonous plant cell.
 14. The plant tissue culture of claim 12,comprising a monocotyledonous plant cell.
 15. The plant tissue cultureof claim 12, comprising a soybean, cotton, maize, or rapeseed plantcell.
 16. A transgenic plant transformed with the DNA construct of claim5.
 17. The transgenic plant of claim 16, wherein the plant is adicotyledonous plant.
 18. The transgenic plant of claim 16, wherein theplant is a monocotyledonous plant.
 19. The transgenic plant of claim 16,wherein the plant is a soybean, cotton, maize or rapeseed plant.
 20. Amethod for controlling weed growth in a crop growing environmentcomprising a plant of claim 16 or a seed thereof, comprising applying tothe crop growing environment an amount of dicamba herbicide effective tocontrol weed growth.
 21. The method of claim 20, wherein the dicambaherbicide is applied over the top to the crop growing environment. 22.The method of claim 20, wherein the amount of dicamba herbicide does notdamage said plant or seed thereof and damages a plant of the samegenotype as the plant, but lacking the construct.
 23. A method forproducing food, feed, or an industrial product comprising: a) obtainingthe plant of claim 16 or a part thereof; and b) preparing the food,feed, fiber, or industrial product from the plant or part thereof. 24.The method of claim 23, wherein the food or feed is grain, meal, oil,starch, flour, or protein.
 25. The method of claim 23, wherein theindustrial product is biofuel, fiber, industrial chemicals, apharmaceutical, or nutraceutical.
 26. A method for producing a dicambatolerant plant comprising introducing the construct of claim 10 into aplant cell and regenerating a plant therefrom that comprises theconstruct of claim
 5. 27. The method of claim 26, further comprisingproducing a dicamba tolerant plant by crossing a parent plant withitself or with a second plant, wherein the parent plant and/or thesecond plant comprises the DNA construct and the dicamba tolerant plantinherits the DNA construct from said parent plant and/or the secondplant.
 28. A method for improving standability of a monocot plantcomprising: a) growing a plant or seed produced by the method of claim27; and b) treating the plant or seed with dicamba.
 29. The method ofclaim 28, further comprising: c) measuring a standability-relatedparameter selected from the group consisting of brace root number,shape, length, or structure; percent lodging; and yield.
 30. A methodfor expressing dicamba monooxygenase in a plant cell comprising growinga plant comprising a nucleic acid construct comprising a nucleotidesequence encoding a small subunit chloroplast transit peptide (CTP) ofSEQ ID NO:4 operably linked to a nucleotide sequence encoding dicambamonooxygenase, thereby expressing the dicamba monooxygenase.
 31. Adicamba tolerant seed for providing protection against pre emergenceapplication of dicamba comprising a DNA encoding chloroplast transitpeptide operably linked to a DNA encoding dicamba monooxygenase, whereinthe DNA encodes a chloroplast transit peptide comprising SEQ ID NO:4.32. The dicamba tolerant seed of claim 31, wherein the DNA encoding thechloroplast transit peptide comprises SEQ ID NO:
 15. 33. The dicambatolerant seed of claim 31, wherein the DNA encodes dicamba monooxygenasecomprising a sequence selected from the group consisting of SEQ ID NOS:26, 28, 32, 34, 36, 38, and 40.